To delineate specific strains at the subspecies level, a variety of bacterial genotyping methods have been developed, including pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) sequencing, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR), multilocus sequence typing (MLST) and ribotyping. Each of these has certain advantages and disadvantages with respect to assessing bacterial clonality.
PFGE is a technique that relies on digestion of the entire bacterial genome by rare-cutting restriction endonucleases followed by separation of the resulting large DNA fragments in an agarose gel subjected to pulsed-field electrophoresis. This method can separate large DNA fragments (of 5 to 10 Mbp) in a size-dependent manner, with relatively few bands to compare (Unemo et al., Clin Microbiol Rev. 24: 447-458, 2011). One advantage of PFGE lies in its high discriminatory power (Hansen et al., Clin Microbiol Infect 8, 397-404, 2002), but PFGE is technically difficult and can result in intralab variability in the absence of careful coordination and planning. PFGE can detect chromosomal rearrangements, caused, for example, by mobile elements in the genome and rapid evolutionary rates. In contrast, MLST is more appropriate for strain phylogeny and large-scale epidemiology (Vimont et al., J Med Microbiol 57:1308-1310, 2008).
PCR-based DNA fingerprinting relies on the principle that the primers bind to specific regions of the DNA, and when this binding occurs in the proper orientation and within an optimum distance, species- or strain-specific amplification products may be generated. Primers such as REP1R-Dt and REP2-Dt, which are derived from the repetitive extragenic palindromic (REP) sequences found primarily in gram-negative bacteria have been used. BOX-PCR is a fingerprinting technique based on the BOX dispersed-repeat motif (e.g., using BOXA1R and BOXA2R primers) that are interspersed throughout the bacterial genome. BOX repeats were first identified in Streptococcus pneumoniae, but are present in a number of bacterial species. (see e.g., Brusetti et al., BMC Microbiology 8:220-, 2008).
In MLST analysis, multiple genes (loci), typically internal fragments of chromosomal housekeeping genes, are sequenced to measure genetic relatedness and analyze sequence variation between alleles from many strains (Maiden, et al. Proc. Natl. Acad. Sci. USA 95:3140-3145, 1998). The DNA sequences useful in MLST are generally conserved, slow to evolve, and, ideally, distributed throughout the genome. MLST is able to characterize the sequence type of each isolate and the genetic relatedness of isolates can be presented as a dendrogram constructed by using the matrix of pairwise differences between the allelic profiles of the genes analyzed. MLST has gained increasing popularity during the last 15 years with >80 MLST schemes being developed for bacterial species important in human infection. However, this method is limited by the cost and labor involved in amplifying, sequencing, editing and concatenating multiple housekeeping genes. In certain instances, PFGE has been found to be more reliable a strain predictor than MLST, while in other strains, MLST is more reliable (Nemoy et al., J Clin Microbiol. 43(4):1776-1781, 2005).
Ribotyping has also recently been developed in an effort to better categorize bacteria species and strains. A ribosomal operon generally consists of the three genes encoding the structural rRNA molecules, 16S, 23S, and 5S, cotranscribed as a polycistronic operon. The copy numbers, overall ribosomal operon sizes, nucleotide sequences, and secondary structures of the three rRNA genes are highly conserved within a bacterial species, with the 16S rRNA being the most conserved. Therefore, 16S rRNA gene sequencing has recently become popular for identification and taxonomic classification of bacterial species (Bouchet et al., Clin Microbiol Rev. 21(2): 262-273, 2008; Kolbert, et al., Curr. Opin. Microbiol. 2:299-305, 1999).
Ribotyping is based on restriction endonuclease cleavage of total genomic DNA followed by electrophoretic separation, Southern blot transfer, and hybridization of transferred DNA fragments with a radiolabeled ribosomal operon probe. Only those bands containing a portion of the ribosomal operon are visualized. The number of fragments generated by ribotyping is a reflection of the multiplicity of rRNA operons present in a bacterial species. Copy numbers of rRNA operons have been found to range from 1 (e.g., for Chlamydia trachomatis) to 15 (e.g., for Photobacterium profundum) (Bouchet et al, supra). The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. See rrndb: the Ribosomal RNA Operon Copy Number Database, Klappenbach et al., Nucl. Acids Res. 29:181-184, 2001.
While the 16S ribosomal subunit gene (16S rRNA) has been widely used to identify bacteria to the species level, this locus is not universally capable of distinguishing all species in a given genus. Multilocus sequencing analyses, often including a portion of the 16S rRNA gene, can enable species assignment when a single gene does not possess sufficient discriminatory power.
WO 2000/008138 describes an rRNA operon alterable bacterium useful for the selection of antibiotics against pathogenic microorganisms. European Patent EP0424473 describes a method of interrupting the expression of a macromolecular synthesis operon in bacteria comprising the step of binding an antisense oligonucleotide to a single stranded DNA or to a mRNA transcribed from the macromolecular synthesis operon. Klappenbach et al., (Appl Environ Microbiol. 66(4):1328-1333, 2000) discuss that no phenotype has been consistently associated with rRNA gene copy number and discloses that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability.
For each bacterial identification technique previously studied, there seem to be strain specific preferences for which method is more effective at differentiating isolates of a particular bacteria, and no one method emerges as the leading method for characterizing bacterial isolates.